Full text of "USPTO Patents Application 10687850"

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UK Patent Application »GB ,,,,2295 676 ra A

(43) Date of A Publication 05.06.1996

(21)

Application No 9520047.3

(51)

INT CL 6

(jOIN 2//0O , liUln iiltt

(22)

Date of Filing 02.10.1995

(52)

UK a (Edition O I

(30)

Priority Data

G1N NBMF NBMK N25B N25B3X N25E1 N25F7B

(31) 9419882 (32) 03.10.1994 (33) GB

U1SS2H0

(56)

Documents Cited

(71>

Applicartt(s)

GB 1585067 A GB 1398947 A EP 0018419 A1

Glasgow Caledonian University Company limited

US 5180968 A US 5138264 A US 4814281 A

US 4262253 A

(Incorporated in the United Kingdom)

(58)

Re Id of Search

2 BIythswood Square, GLASGOW, G2 4AD,

UK CL (Edition N ) GIN NBCC NBMF NBMK NBMX

United Kingdom

INTCL 6 G01N 27/06 27/08 27/10, G01R 27/22

Online :WPI

(72)

Inventor(s)

Arthur McNaughtan

(74)

Agent and/or Address for Service

Cruikshank & Fairweather

19 Royal Exchange Square, GLASGOW. G1 3AE,

United Kingdom

(54) Conductivity measuring system comparing two detection channels

(57) The system comprises two detection chsnnels 11, 12, the first of which receives a mobile phase plus the
analyte and the second of which receives only the mobile phase. Each channel has a detector 11,12 comprising
a microelectrode and a reference electrode for immersion In the received solution. An ac modulated voltage is
applied across each pair of electrodes and the resulting current measured. Phase sensitive detection means 20
are provided for determining the faradic component of the current flowing between each pair of electrodes. The
system further comprises means 21 for differentially combining the faradic components of the two channels
and amplifier 22 for balancing the channels when mobile phase only is applied to both channels.

At least one drawing originally filed was informal and the print reproduced here is taken from a later filed formal copy.
The print reflects an assignment of the application under the provisions of Section 30 of the Patents Act 1977.

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KTiRCTROCHEMI CAL DET ECTION SYSTEMS

The present invention relates to electrochemical
detection systems and in particular, though not
necessarily, to electrochemical detection systems for use
in high performance liquid chromatography.

The use of amperometric sensors to determine the
concentration of an ionised analyte present in a solution
is widespread. Such sensors rely upon the dependence of
faradaic current across an electrode/electrolyte- boundary
on the concentration of ions in the electrolyte. As the
ionic concentration of the electrolyte increases, for a
given voltage applied between a pair of electrodes, the
faradaic current will tend to increase. In order to
estimate amperometrically the concentration of a specific
analyte in a solution however, it is generally necessary
to first purify the solution to a point where it contains
substantially only the analyte under investigation.

A commonly used purification technique is that known
as high performance liquid chromatography (HPLC) which
involves passing an unpurified solution, containing the
analyte of interest, under pressure through a column packed
with very fine polymer beads (for example beads sold under
the trade name "Sophadex") . The rate at which particular
components of the unpurified solution flow through the
column depends upon the size of the component and the
relative porosity of the column filling. Components having
different sizes will flow through the column at different
rates and the output from the foot of the column will be

a series of fractions containing different ones of the
solution components. HPLC systems may be further refined
by adding for example positive or negative charges to the
beads to inhibit or advance the flow of certain components
through the column. In general, HPLC systems require to
be calibrated by running various 'pure' samples through the
column to determine the flow rate of specific components.

Amperometric sensing techniques are used to estimate
the concentration of an analyte of interest contained in
a sample purified using HPLC. Whilst this technique has
proved useful, conventional electrochemical detection
systems offer a relatively limited detection range due
primarily to noise. Noise arises due to a number of
factors including electromagnetic interference and
contributions to the electrode/electrolyte current by
phenomenon other than the faradaic effect. Additional
problems with conventional electrochemical detection
systems include the relatively long time required to
establish a steady state current across the
electrode/electrolyte interface and the relatively low
resistivity support solutions which must be used in order
to maintain adequately high currents (to ensure a
sufficiently high signal to noise ratio). In addition, the
manufacturer of HPLC systems with integrated
electrochemical detection systems is difficult due to the
relatively large size of existing electrochemical detection
systems .

It is an object of the present invention to overcome

or at least mitigate certain of the disadvantages of
conventional electrochemical detection systems. In
particular, it is an object of the present invention to
provide a low noise, highly sensitive microelectrode
electrochemical detection system which can be integrated
into an HPLC system.

According to a first aspect of the present invention
there is provided an electrochemical detection system for
use in determining the concentration of an analyte in
solution, the system comprising at least two detection
channels, each channel comprising:

first and second electrodes for immersion in a
solution;

means for applying a voltage across said electrodes
to cause a current to flow therebetween when the electrodes
are immersed;

means for monitoring the flow of current between the
electrodes when said voltage is applied; and

phase sensitive detection means for determining the
f aradic component of a current flowing between the
electrodes,

wherein the electrodes of a first of the channels are
arranged to be immersed in said solution in the absence of
the analyte and the electrodes of the second channel are
arranged to be immersed in said sample solution containing
the analyte, the system further comprising means for
differentially combining the faradic components of the two
channels.

The provision of an additional reference channel and
a differential output enable background noise common to
both channels, e.g. electromagnetic interference or
electrode currents resulting from the conductivity or
electroactivity of the mobile phase (i.e. the solution in
which the analyte is dissolved) , to be substantially
reduced. The use of phase sensitive detection means
additionally enables the effect of electrode double layer
capacitance to be substantially reduced.

Preferably for each channel at least one of the two
electrodes is a microelectrode having a surface area of
less than O.Olinm 2 and preferably less than 0,0025mm 2 . The
use of microelectrodes enables a reduction in the current
flowing across the electrode/electrolyte boundary which in
turn enables the use of mobile phases, in which the analyte
is dissolved, having higher resistivities. The use of
microelectrodes also reduces the time required to establish
a steady state current across the electrode electrolyte
interface.

Preferably, the means for applying a bias voltage
across the two electrodes of each channel comprises means
for applying a dc bias voltage, modulated with a relatively
low voltage ac signal, across the electrodes. The bias
voltage means may be arranged to operate in a pulsed
amperometric detection mode. Preferably, the same voltage
is applied across both electrodes from a common voltage
source.

Preferably, the phase sensitive detection means of

each channel comprises a lock-in amplifier which receives
as its reference signal the electrode a.c. bias voltage.
The gain of the lock-in amplifier of one or both channels
may be adjusted during a set-up stage, during which both
channels receive only the mobile phase, to null the output
of the differential combining means* Alternatively, a null
setting may be achieved by incorporating a separate
variable gain amplifier into one of the channels and
varying the gain of that amplifier during the set-up stage.

Preferably, the differential combining means comprises
a differential amplifier which provides at its output a
signal proportional to the difference between the outputs
of the two lock- in amplifiers.

According to a second aspect of the present invention
there is provided an HPLC system for determining the
concentration of a component of a sample solution, the
system comprising an electrochemical detection system
according to the above first aspect of the invention.

For a better understanding of the present invention
and in order to show how the same may be carried into
effect an embodiment of the invention will now be described
with reference to the accompanying drawings, in which:

Figure 1 illustrates schematically a high performance
liquid chromatography system incorporating an
electrochemical detection system embodying the present
invention;

Figure 2 shows an electrochemical detector of the
system of Figure 1;

Figure 3 illustrates an equivalent circuit for an
electrode/electrolyte interface;

Figure 4 shows schematically a circuit for
implementing the electrochemical detection system of Figure
5 1;

Figure 5 illustrates a typical output signal from the
circuit of Figure 4;

Figure 6 shows schematically an alternative embodiment
of the present invention.

10 There is shown in Figure 1 a high performance liquid

chromatography (HPLC) system 1 having a column 2 which is
filled with an appropriate flow retarding filler as
described above. A mobile phase reservoir 3 contains a
mobile phase supply, which can be water or another solvent.

15 The reservoir is coupled to the column 2 via a pump 8 and
an injection valve 9 which maintain the required high
pressure within the column. The analyte is injected into
the mobile phase at the injection valve 9. The output 10
from the foot of the column is supplied to a detector 11

20 (EDS) which will be described hereinbelow.

The pump output is also coupled to a second detector
12 via a pressure regulator valve 13. In operation, during
an initial set-up stage the reservoir 3 supplies only the
mobile phase which is in turn supplied to the two detectors

25 via respective valves (the detectors being at a lower
pressure than the column) . Subsequently, the valve feeding
the mobile phase to the second detector remains open and
the analyte is injected into the column 2 via the injection

valve 9 to supply the mobile phase containing the injected
analyte to the first detector.

Figure 2 shows in more detail the arrangement of the
detectors 11,12 of the EDS of Figure 1 (both arrangements
being substantially the same) . The detectors comprise a
microelectrode 14 which comprises the exposed end face of
a platinum wire 15 (or other suitable material such as
gold) extending through an insulating glass or plastic tube
16. Methods of producing such microelectrodes are well
known. The detectors are also provided with a reference,
or return, electrode 17 which has a relatively large
surface area compared to the microelectrode (e.g. 10 to 100
times). The reference electrode may be of any suitable
material although silver/silver chloride electrodes are
preferred due to their relatively low impendence and their
high electrical stability. Both the microelectrode and the
reference electrode are arranged to be immersed in the
solution fed via the pump 8.

The electrodes of each detector are coupled to a
voltage source 18 which is arranged to apply both a dc bias
voltage and a small ac modulating voltage in parallel
across each pair of electrodes. Coupled in series between
the electrodes of each detector and the voltage source 18
is an electrometer operational amplifier 19 , having a
feedback resistor r„ which develops an output voltage
proportional to the current i e flowing between the
microelectrode and the return electrode. The electrometer
operational amplifiers present a very low impedance to the

respective circuits and therefore do not significantly load
these circuits. A lock-in amplifier 20 , to be described
hereinbelow, is connected across each of the electrometer
operational amplifiers 19. Preferably, pulsed amperometric
detection is used in which the voltage is applied across
the electrodes only in short pulses. This helps prevent
fouling of the electrodes.

For each detector ,^ the reference electrode 17 and the
microelectrode 14 present two electrode/electrolyte
interfaces across which current flowing around the circuit
must pass. Both of these interfaces represent complex
impedances in the series circuit although, as impedance is
approximately inversely proportional to the interface
surface area, for the purpose of analysis the impedance of
the reference electrode 17 can be neglected. Figure 3
shows an equivalent circuit of the electrode/ electrolyte
interface presented by the microelectrode 14. The
microelectrode interface can be represented as a
capacitance C D corresponding to the electrode/electrolyte
double layer in parallel with a complex impedance Z F
representing the faradic contribution. Current flowing
across the electrode/electrolyte boundary i e will therefore
comprise a first fraction i D which flows through the double
layer capacitance and a second fraction i F which flows
through the faradic impedance. It is this second fraction
which is analyte concentration dependent and which must be
derived in order to accurately estimate analyte
concentration .

From the equivalent circuit shown in Figure 3, it is
apparent that the double layer current will be phase
shifted by approximately 90° whilst the faradic current
will be shifted by somewhere between 0° and 90°, typically
45°. In order to separate out the faradic component, the
voltage developed by the electrometer operational amplifier

19 is coupled as a measured signal to a lock-in amplifier

20 which also receives as a reference signal the ac
modulating voltage from the voltage source 18 . The lock-in
amplifier provides at its output a signal V^, where

_ signal voltage x ref voltage ^ (ft + x }
v y out ~ 2

where 0 is the phase difference between the two signals and
<t> is an arbitrary phase shift. When 0 = 0, cos 5 will
equal 1/V2 when 9 = 45° and will equal 0 when 0 = 90°. The
lock-in amplifier therefore effectively nulls the double
layer current component i D and provides an output which is
substantially proportional to the faradic current component
i p . Variations in the surface properties of electrodes may
cause the double layer and faradaic currents to be phase
shifted, e.g. to 45° and 22° respectively. These shifts
can be compensated for by adjusting the value of 0 which
can be set in the lock-in amplifier.

Figure 4 shows a circuit arrangement for processing
the outputs provided by the sample and reference detectors
to provide a signal indicative of the concentration of a
component in the solution. As described above, both

detectors are fed by a common voltage source 18 which also
provides the modulated bias voltage to the lock-in
amplifier of each detector.

The compensated output signals are coupled to
respective inputs of a differential amplifier 21 which
provides an output signal proportional to the difference
between the two compensated signals. Assuming that the
electrodes , and other conditions, of both detectors are
identical the effects of noise common to both detectors
will be eliminated.

In practice it is difficult or even impossible to
obtain a perfect match between the detectors, e.g. due to
manufacturing tolerances. However such differences can be
compensated for by carrying out a set-up stage in which
both detectors receive only the mobile phase. The gain of
the output stage of one of the lock-in amplifiers 20 is
then adjusted to null the output of the differential
amplifier 21.

Figure 5 illustrates a typical output of the system
of Figures 1. During the set-up stage, when the detectors
receive only the mobile phase, the output of the
differential amplifier is a substantially constant dc
voltage indicating intrinsic differences between the two
detectors. At a time t,, the gain of one of the lock-in
amplifiers is adjusted to null the output of the
differential amplifier. Subsequently the analyte, which
contains at least three components, is introduced into the
HPLC column. The component which travels fastest (1)

through the column produces a peak in the differential
amplifier output at time ^ whilst the slower travelling
components (2) and (3) produce peaks at times t 3 and t 4
respectively. By precalibrating the detectors with
standard solutions containing ones of the three components,
the concentration of the components in the sample can be
estimated from the amplitude of the peaks.

Figure 6 shows schematically an alternative embodiment
of the present invention in which components already
described with reference to Figures 1,2 and 4 are indicated
with like reference numerals.

Rather than coupling the mobile phase pump 8 to the
second detector 12 via a pressure regulator valve (as shown
in Figure 1), this embodiment has both detectors 11,12
connected to the outlet of the separating column 2 with
approximately 2m of tubing separating the two detectors.
This length is such that by the time the analyte reaches
the second detector 12 it will have passed through the
first detector 11. Similarly, as the analyte passes
through the first detector 11, it will not yet have reached
the second detector 12. Thus the analyte will produce a
pair of spikes of opposite phase at the output of the
differential amplifier 21.

The embodiment of Figure 6 is further modified by the
inclusion of a variable gain amplifier 22 coupled between
the output of one of the lock-in amplifiers 20 and the
differential amplifier 21. This eliminates the need for
the lock-in amplifiers to have provision for varying their

gain and instead the gain of the amplifier 22 can be
adjusted to null the output of the system.

It has been found that noise levels can be reduced
significantly by shielding the electrode connections with
a shield driven by the modulated bias voltage.

It will be appreciated that variations may be made to
the above described embodiment without departing from the
scope of the invention. For example, instead of applying
a continuous or pulsed voltage across the electrodes, a
saw-wave voltage may be applied to enable cyclic
voltammetry to be carried out. The ac modulating voltage
may be, for example, a sinusoidal or a square wave voltage.
An embodiment of the invention may comprise more than two
detection channels with switching means for coupling
selected ones of the channels to the solution reservoir.

CLAIMS

1. An electrochemical detection system for use in
determining the concentration of an analyte in solution,
the system comprising at least two detection channels, each
channel comprising:

first and second electrodes for immersion in a

solution;

means for applying a voltage across said electrodes
to cause a current to flow therebetween when the electrodes
are immersed;

means for monitoring the flow of current between the
electrodes when said voltage is applied; and

phase sensitive detection means for determining the
faradic component of a current flowing between the
electrodes,

2. A system according to claim 1, wherein at least one
of the two electrodes is a microelectrode having a surface
area of less than 0.0025mm 2 .

3. A system according to claim 1 or 2, wherein the means
for applying a bias voltage across the two electrodes of
each channel comprises means for applying a dc bias

voltage, modulated with a low voltage ac signal, across the
electrodes.

4. A system according to claim 3, wherein the same
voltage is applied across both electrode pairs from a

5 common voltage source.

5. A system according to claim 3 or 4, wherein the phase
sensitive detection means of each channel comprises a lock-
in amplifier which receives as its reference signal the
corresponding electrode a.c. bias voltage.

10 6. A system according to claim 5, wherein the gain of the
lock-in amplifier of one or both channels may be adjusted
during a set-up stage, during which both channels receive
only the mobile phase, to null the output of the
differential combining means.

15 7. A system according to claim 5 and comprising a
variable gain amplifier coupled in series with one of the
lock-in amplifiers so that a null setting may be achieved
during a set-up stage, during which both channels receive
only the mobile phase, to null the output of the

20 differential combining means.

8. A system according to any one of the preceding claims,
wherein the differential combining means comprises a
differential amplifier arranged to provide at its output
a signal proportional to the difference between the outputs

25 of the two lock-in amplifiers.

9. An electrochemical detection system substantially as
hereinbefore described with reference to Figures 1 to 5 of
the accompanying drawings or with reference to those

Figures as modified by Figure 6.

10. An HPLC system for determining the concentration of
a component of a sample solution, the system comprising an
electrochemical detection system according to any one of
the preceding claims.

11. An HPLC system substantially as hereinbefore described
with reference to Figures 1 to 5 of the accompanying
drawings or with reference to those Figures as modified by
Figure 6.

Application No:
Claims searched:

GB 9520047.3
1-11

Patent
Office

•6

Examiner:
Date of search:

D J Mobbs

24 November 1995

Patents Act 1977

Search Report under Section 17

Databases searched:

UK Patent Office collections, including GB, EP, WO & US patent specifications, in:
UK CI (Ed.N): GIN NBCC, NBMF, NBMK, NBMX.
lnt CI (Ed.6): G01N 27/06, 27/08, 27/10; G01R 27/22.
Other: ONLINE: WPI. .

uutum

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Full text of "USPTO Patents Application 10687850"

See other formats